Role of Mobile Zinc in Neurobiology
Zinc is the second most abundant metal ion in living systems. Its biological importance is accentuated by the fact that approximately 10% of the human genome is dedicated to the zinc proteome. Whereas the majority of zinc is highly regulated and tightly bound within protein scaffolds, a growing body of evidence suggests the presence of readily exchangeable or “mobile” zinc (“mZn”) located within the pancreas, prostate, and brain. The importance of mZn in human health has been extensively documented, but knowledge of its physiology and pathology is incomplete. Taylor, C. G. Biometals 2005, 18, 305; et al. Mol Cancer 2006, 5, 17; Frederickson, C. J. et al. Nat Rev Neurosci 2005, 6, 449.
Fluorescent-based probes are the most common agents utilized to image mobile zinc within cellular environments. Que, E. L. et al. Chem Rev 2008, 108, 1517. Broadly speaking, zinc probes are divided into two categories: small molecule (“SM”) sensors; and genetically encoded (“GE”) sensors. SM-based probes offer a scaffold that is readily modified, providing sensors with chemical and physical properties that can be tuned for specific applications. On occasion SM probes can also display unpredictable subcellular distribution, which has led to controversy and confusion in biological communities. Kay, A. R. Trends Neurosci 2006, 29, 200. Conversely, GE sensors offer impressive control over the subcellular localization of the probe and are inherently biocompatible. Yet, GE probes suffer from the limited tunability of their metal-binding motifs, large sizes, and requirement of complex procedures for their incorporation into mammalian cells.
Metal ions are essential reactive cofactors, obligatory for carrying out complex chemical processes vital to cell metabolism. Yet, the reactive nature of metal ions requires tight regulation of their concentrations and cellular distribution. When unregulated, mobile metal ions have been implicated in multiple neurological disorders including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Frederickson, C. J. et al. Nat Rev Neurosci 2005, 6, 449.
In this context, it is surprising that large concentrations of mZn (˜0.5 mM) occur in regions of the brain containing neuron cell bodies. Que, E. L. et al. Chem Rev 2008, 108, 1517. Although most of this zinc is “static”—i.e., tightly associated with a protein scaffold and serving both as structural and functional components in protein biochemistry—the existence of pools of mZn implies a functional role in neurological biochemistry. Bush, A. I. Curr Opin Chem Biol 2000, 4, 184. Zinc levels in the brain are non-uniformly distributed, with high concentrations occurring in the hippocampus, amygdala, and olfactory bulb. Id.
Recently, observations from the mossy fiber (mf) axons in the CA3 region of the hippocampus—an area of the brain responsible for learning and memory—suggested that the underlying mechanism behind zinc neurochemistry is both complex and nuanced. Zinc released from glutamatergic synaptic vesicles has been proposed to associate with zinc permeable gated channels, including N-methyl-D-aspartate (NMDA) receptors, voltage-gated calcium channels, and the calcium-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic)/kainite channel (Ca2+-A/K), where it can enter postsynaptic axons and/or function to inhibit postsynaptic mossy fiber long-term potentiation (mf-LTP). Frederickson, C. J. et al. Nat Rev Neurosci 2005, 6, 449; Pan, E. et al. Neuron 2011, 71, 1116. Portions of the released zinc have also been proposed to “re-enter” presynaptic termini through calcium-gated ion channels. Upon re-entry, zinc can transactivate the tyrosine kinase receptor (TrkB), independent of neurotrophins, initializing a chain of molecular events critical to both presynaptic mf-LTP and neuronal plasticity. Pan, E. et al. Neuron 2011, 71, 1116; Huang, Y. Z. et al. Neuron 2008, 57, 546. This “dual action” of vesicular zinc appears to be critical in regulating the effectiveness of mf-CA3 synapses and ensuring proper hippocampal function in health and disease, but the mechanistic details of its actions remain incomplete and highly debated.
Imaging Mobile Zinc
Divalent zinc is a good Lewis acid, redox inactive under physiological conditions, and able to adopt multiple binding geometries. Its d10 closed-shell electronic structure renders zinc spectroscopically silent, complicating noninvasive in vivo imaging. Currently, fluorescent probes provide the most facile way to image zinc within a cellular environment. Tomat, E. et al. Curr Opin Chem Biol 2010, 14, 225.
One such family of zinc probes is the ZinPyr (ZP) family of sensors first reported by in 2000. Walkup, G. K. et al. J Am Chem Soc 2000, 122, 5644. ZP1 is based on a fluorescein platform that is further modified with two dipicolylamine (DPA) “metal-binding arms”, which function to quench fluorescein emissions via photoinduced electron transfer (PET) in the metal-free form. Upon coordination to zinc, the electronic configuration of ZP1 changes, resulting in an attenuation of the PET effect and recovery of fluorescein emissive properties. ZP1 has several attributes that make it a model probe: (1) it is formed in a high yielding, “one-pot”, Mannich reaction between 2′,7′-dichlorofluorescein and the iminium ion condensation product of formaldehyde and DPA; (2) it is a zinc selective “turn on” sensor, meaning it is non-responsive to the presence of other biologically relevant metal ions such as Ca2+, Mg2+, Mn2+, Cu2+, or Fe2+; and (3) it is excitable with visible (˜500 nm) light, making it compatible with the 488 nm argon line of most fluorescence microscopes, as well as reducing background fluorescence attributed to biological auto-fluorescence. To date, a library of sensors in the ZP family, and an extensive group of related ZS, QZ and ZPP derivatives have been synthesized, allowing access to probes with a wide range of zinc-binding affinities and dynamic ranges. Nolan, E. M. et al. Acc Chem Res 2009, 42, 193; Pluth, M. D. et al. Annu Rev Biochem 2011, 80, 333. The ZP family of probes has been well documented to function in cellular systems, such as HeLa, hippocampal slices, and pancreatic β-insulinoma cells, and even in live animals (TRAMP—a mouse model of prostate cancer), demonstrating their practical utility in addressing biological questions. Nolan, E. M. et al. Acc Chem Res 2009, 42, 193; Ghosh, S. K. et al. Cancer Res 2010, 70, 6119.
Proteins and Peptides as Zinc Sensor Scaffolds
Proteins are among the most versatile biomolecules. Their ability to arrange chemically diverse functional groups in a three dimensional architecture enables them to carry out complex chemical transformations with a reactivity and selectivity unparalleled in synthetic systems. Moreover, the large and relatively featureless surface of most proteins necessitated the evolution of numerous weak but additive interactions conferring the ability to form tight and specific connections with other biomolecules, such as cell receptors, nucleic acids, and cofactors, even within the crowded cellular environment. This biological specificity has led to the use of proteins and peptides as therapeutics, drug delivery vehicles, and metal sensors. Previously, development of peptide-based zinc sensors mainly involved the reengineering of small metal-binding proteins, most notably the zinc finger fold. Godwin, H. A. et al. J Am Chem Soc 1996, 118, 6514; Shults, M. D. et al. J Am Chem Soc 2003, 125, 10591; Walkup, G. K. et al. J Am Chem Soc 1996, 118, 3053; Walkup, G. K.; Imperiali, B. J Am Chem Soc 1997, 119, 3443. This seminal work invigorated the nascent field of peptide-based biosensors by highlighting the way natural and/or non-natural components could be “mixed-and-matched,” achieving sensors that retained biological activity and specificity while reporting on targeted analytes. From a zinc sensor perspective, these probes were limited by the UV excitation of the fluorophores, narrow range of metal-binding affinities, or metal-induced self-assembly/aggregation of the sensor. In other words, these characteristics prevented their wider use and acceptance in biological communities.
The introduction of green fluorescent protein (GFP) variants has led to the creation of GE metal-sensor proteins. Metal-sensor proteins can readily be made ratiometric by joining photophysically complementary GFP variants together with a short peptide sequence that mimics the metal-binding region of a natural metal-binding protein (e.g., Atox1, MTIIa, and Zif268). GE sensors have been successfully applied intracellularly to measure the concentration and spatial distribution of mZn, as well as subcellularly, to determine mZn levels in the ER and Golgi. Dittmer, P. J. et al. J Biol Chem 2009, 284, 16289; Vinkenborg, J. L. et al. Nat Methods 2009, 6, 737; Qin, Y. et al. Proc Natl Acad Sci USA 2011, 108, 7351. Although GE probes obviate complications surrounding probe internalization, subcellular localization, and biological compatibility, they have narrow wavelength availability, relatively low photochemical stability, and limited chemical diversity. Pazos, E. et al. Chem Soc Rev 2009, 38, 3348.
There exists a need for a new class of zinc sensors that couple the chemical and physical characteristics of small-molecule based probes with the biological specificity of peptide scaffolds.